Immunotherapies, particularly those that inhibit the PD1/PD-L1 interaction and drive T cells to recognize and kill tumor cells, have shown striking responses in a small subset of late stage, treatment refractory squamous lung tumors. However, the majority of patients do not have a lasting response. One way to boost response is to include an epigenetic inhibitor, such as one targeting the Polycomb Repressive Complex 2 (PRC2), to influence the tumor cell and microenvironment heterogeneity. My central hypothesis is that EZH2 inhibitors will boost immunotherapy response in squamous tumors, both by increasing the PD-L1 expressing tumor propagating cells (TPCs) and by depleting immunosuppressive tumor associated neutrophils. The overarching goal of the proposed study is to validate combining EZH2 inhibition with PD1/PD-L1 targeted immunotherapy and learn the molecular mechanisms when the treatment is successful as well as when it is not successful. In Aim 1, I will use both mouse models and human patient derived organoid cultures, and serial orthotopic transplantation to examine NGFR and PD-L1 expression and tumor propagation abilities of lung SCC cells after EZH2 inhibitor treatment. I will also test if non-TPC cells can de-differentiate and continue to fuel tumor growth by sorting for presence or absence of the putative TPC cell surface marker nerve growth factor receptor (NGFR). The epigenetic and transcriptional consequences of EZH2 inhibition on squamous lung tumor cells will be assessed by ChIP-seq and RNA-seq. In Aim 2, I will focus on the tumor associated neutrophils (TANs). I will compare TANs from placebo treated mice to those from EZH2 inhibitor treated mice for abundance, migration capacity and ability to suppress T cells. I will also use an EZH2 conditional knock-out mouse model to further characterize how EZH2 loss affects TANs. Again ChIP-seq and RNA-seq will be used to dissect the molecular changes driven by EZH2 inhibition in the neutrophil populations. In Aim 3, I will treat immune-competent squamous lung cancer bearing mice with the EZH2 inhibitor GSK126 or EPZ-6438 (Tazemetostat), and the immunotherapy PD-1 antibody and follow tumor growth by magnetic resonance imaging. I will characterize tumor phenotypes in responders and non-responders and examine tumors that develop acquired resistance to this therapeutic drug combination. Completion of these aims will solidify the efficacy of a promising therapeutic combination and uncover mechanisms by which tumor hierarchies and microenvironments are changed by EZH2 inhibitors in squamous lung cancers. Given that one arm of a Phase 1/2 clinical trial combining EZH2 inhibition with anti-PDL1 just began recruiting late stage non-small cell lung cancer patients, learning the phenotypes and mechanisms of responders and non-responders will be extremely timely for any Phase 2/3 trials that ensue.